The overall objective of this project is to understand the cellular and molecular mechanisms responsible for the specification, patterning, and differentiation of internal organs during development. More specifically, how the elaborate network of blood vessels arises during vertebrate embryogenesis. The regulation of blood vessel formation is currently a subject of intense research study, with profound potential implications for human health. Antiangiogenic and proangiogenic therapies show enormous promise for combating cancer and treating limb or cardiac ischemia, respectively. Efforts to develop these therapies, as well as targeted treatments for atherosclerosis, depend on a detailed understanding of the genetic mechanisms of normal blood vessel formation, about which relatively little is still known, and on the identification of new molecular therapeutic targets. The zebrafish, a small tropical freshwater fish, possesses a unique combination of features that make it particularly well suited for these goals. The fish is a genetically tractable vertebrate with a physically accessible, optically clear embryo. These features provide a tremendous advantage for studying vascular development, because they permit direct, noninvasive observation of every blood vessel in a living embryo throughout its development and the isolation of mutants that cause defects in the formation of embryonic blood vessels. Major aims of the laboratory include: (i) studying the phenotypic and molecular basis for the defects in vascular patterning mutants, (ii) elucidating the morphogenetic and molecular mechanisms responsible for blood vessel formation in the embryonic trunk, and (iii) developing new experimental tools for studying vascular embryogenesis in the zebrafish. The zebrafish vascular mutants we study include mutants that display disruption of cranial vessel formation, formation of abnormal arterial-venous connections, and localized blockage of circulation. By positional cloning of the gridlock and violet beauregarde vascular patterning utants, we have shown that these mutants are due to defects in a novel hairy-related bHLH factor expressed specifically in the dorsal aorta and in a zebrafish ortholog of the TGF-beta superfamily type 2 receptor ALK1, respectively. Interestingly, defects in the human ALK1 gene are responsible for Hemorrhagic Telangiectasia type 2 (HHT). HHT is a hereditary vascular disorder characterized by arterial-venous malformations that lead to a high incdence of hemorrhage and stroke. Further analysis of these and other mutants and genes is in progress. We have also obtained novel insights into molecular and morphogenetic mechanisms of trunk blood vessel formation. These insights include our recent discovery of previously unknown roles for two well-known signaling pathways, the Hedgehog and Notch pathways, in the specification and arterial-venous differentiation of developing blood vessels. By examining Hedgehog pathway mutants and experimentally manipulating Hedgehog signaling in vivo, we have found that Sonic Hedgehog (Shh) signaling is necessary for specification of the trunk dorsal aorta. In the absence of Shh signaling the dorsal aorta fails to form, while ectopic activation of Shh signaling leads to apparent ?arterialization? of venous endothelial cells. We have used similar genetic and experimental methods to activate or repress Notch signaling in developing zebrafish embryos. Notch signaling is not necessary for specification of either the dorsal aorta or posterior cardinal vein, but it is essential for proper arterial-venous differentiation of these and other embryonic blood vessels. In order to further exploit the advantages of the zebrafish , an important additional aim of this project has been to develop new experimental tools for studying blood vessel formation in this organism. Using a novel confocal microangiographic technique we devised, we have prepared a detailed three-dimensional atlas of the complete vascular circuitry of the zebrafish embryo and early larva. An interactive, online version of this atlas is available at: http://mgchd1.nichd.nih.gov:8000/zfatlas/Intro%20Page/intro1.html. We have also generated transgenic zebrafish lines expressing green fluorescent protein (GFP) in blood vessels, making it possible for us to visualize the blood vessel formation in intact, living embryos. We have developed methodologies for long-term multiphoton confocal timlapse imaging of the dynamics of blood vessel formation in these transgenic zebrafish. Using these methods, we have made a number of unexpected observations about the morphogenetic mechanisms of blood vessel formation in developing animals. These include the elucidation of a novel, two-step mechanism for formation and interconnection of the intersegmental vessels of the trunk. Taken together, our findings underscore both the complexity of mechanisms guiding embryonic blood vessel formation and the power of the zebrafish for dissecting these mechanisms.